This invention relates to processes for oligonucleotide synthesis and purification. In particular, this invention relates to the removal of the 5xe2x80x2-terminal dimethoxytrityl sugar-protecting groups following oligonucleotide synthesis. This invention is amenable to the purification of oligonucleotides following large-scale synthesis.
Oligonucleotides and their analogs are routinely used in many diagnostic and research applications, as probes, primers, linkers, adaptors and antisense oligonucleotides. Antisense oligonucleotides have been used routinely in research to study the functions of gene products, i.e. proteins, by modulating the expression thereof. These oligonucleotides are designed to bind in a specific fashion to a particular mRNA sequence by hybridization (i.e., oligonucleotides that are specifically hybridizable with a target mRNA). Such oligonucleotides and oligonucleotide analogs are intended to inhibit the activity of the selected mRNA by any of a number of mechanisms, i.e. to interfere with translation reactions by which proteins coded by the mRNA are produced or initiate RNase H degradation of the mRNA.
The inhibition of the formation of the specific proteins that are coded for by the mRNA sequences allows the study of functions of certain genes.
The specificity of antisense oligonucleotides and their analogs are also used therapeutically. Their mechanism of action limits side effects while increasing specificity. Presently, there are numerous antisense oligonucleotides in clinical trials against a wide range of targets and diseases and recently the first antisense oligonucleotide was approved by the FDA for marketing.
Applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities. This is especially true for their use as commercially available pharmaceutical drugs. The large-scale synthesis and purification of oligonucleotides on an economic scale presents different challenges than those in synthesis of small amounts for research.
Synthesis of oligonucleotides can be accomplished using both solution phase and solid phase methods. A general review of solid-phase versus solution-phase oligonucleotide synthesis is given in the background section of Urdea, et al. U.S. Pat. No. 4,517,338, entitled xe2x80x9cMultiple Reactor System And Method For Oligonucleotide Synthesisxe2x80x9d. Oligonucleotide synthesis via solution phase can be accomplished with several coupling mechanisms.
One such solution phase preparation utilizes phosphorus triesters. Yau, E. K., et al., Tetrahedron Letters, 1990, 31, 1953, report the use of phosphorous triesters to prepare thymidine dinucleoside and thymidine dinucleotide phosphorodithioates. However, solution phase chemistry requires purification after each internucleotide coupling, which is labor intensive and time consuming.
Further details of methods useful for preparing oligonucleotides may be found in Sekine, M., et. al., J. Org. Chem., 1979, 44, 2325; Dahl, O., Sulfur Reports, 1991, 11, 167-192; Kresse, J., et al., Nucleic Acids Res., 1975, 2, 1-9; Eckstein, F., Ann. Rev. Biochem., 1985, 54, 367-402; and Yau, E. K. U.S. Pat. No. 5,210,264.
The current method of choice for the preparation of naturally occurring oligonucleotides, as well as oligonucleotides with modified internucleotide linkages such as phosphorothioate and phosphoro-dithioate oligonucleotides, is via solid-phase synthesis wherein an oligonucleotide is prepared on a polymer support (a solid support).
Solid-phase synthesis relies on sequential addition of nucleotides to one end of a growing oligonucleotide chain. Typically, the 3xe2x80x2-most nucleoside (having protecting groups on any exocyclic amine functionalities present) is attached to an appropriate solid support and activated phosphorus compounds (typically nucleotide phosphoramidites, also bearing appropriate protecting groups) are added stepwise in a 3xe2x80x2 to 5xe2x80x2 direction to elongate the growing oligonucleotide. The activated phosphorus compounds are reacted with the growing oligonucleotide using xe2x80x9cfluidized bedxe2x80x9d technology to mix the reagents. A number of solid-phase synthesizers are available commercially which automate this process.
A common requirement for oligonucleotide synthesis, whether by solution phase or solid phase methods, is protection of the 5xe2x80x2-OH group of the incoming nucleoside or nucleotide monomer. The internucleoside linkages are formed between the 3xe2x80x2-functional group of the incoming nucleoside and the 5xe2x80x2-OH group of the 5xe2x80x2-most nucleoside of the growing, support-bound oligonucleotide. Many methods of oligonucleotide synthesis require the phosphorylation or phosphitylation of the 3xe2x80x2-OH, and thus, a temporary protecting group is necessary on the 5xe2x80x2-OH (Gait, M. S., Oligonucleotide Synthesis A Practical Approach, IRL Press 1985, 1-22). A 5xe2x80x2-OH protecting group is desired to prevent dimerization of the incoming nucleosides . The 5xe2x80x2-OH protecting group needs to be very acid labile to prevent depurination of the oligonucleotide during removal of the protecting group. The most common agent is dimethoxytrityl (DMTr).
In practice, there are two steps where DMTr is required. During the stepwise synthesis of oligonucleotide, a DMTr protected monomer is added to the elongating chain. The trityl group is removed from the 5xe2x80x2-most nucleotide during a specific detritylation step, most often using a solution of a mild organic acid such as dichloracetic acid or trichloroacetic acid in an organic solvent (e.g. toluene or dichloromethane). After completion of oligonucleotide synthesis and cleavage from the solid support, the 5xe2x80x2-terminal DMTr group is kept on the oligonucleotide (referred to as a DMTr-on oligonucleotide) to facilitate separation from side reaction products which do not have DMTr. High performance liquid chromatography is often used for this purification step. After this initial purification, the final trityl needs to be removed giving a DMTr-off oligonucleotide. Due to the less stringent requirement for an anhydrous environment, a weak acid such as glacial acetic acid (or a dilute solution thereof) can be used for detritylation of the final product.
In a large-scale synthesis method described by Beaucage, S. (in Chapter 3 of Protocols for Oligonucleotides and Analogs, Agrawal, S. (Ed.), 1993, Humana Press, Totowa, N.J.), after reverse-phase HPLC purification, fractions containing the product of interest are pooled. The fractions containing oligonucleotide are typically in methanol and a salt. Triethylammonium acetate is the most common salt for small scale synthesis of oligonucleotides. The solvent is removed using rotary evaporation, leaving the DMTr-on oligonucleotide. The DMTr-on oligonucleotide is treated with 80% glacial acetic acid typically for 30 or 60 minutes. The oligonucleotide is then recovered by ethanol precipitation and applied to a PD-10 Sephadex(copyright) G-25 column to recover the sodium salt of the oligonucleotide. Alternatively, Padmiapriya, A. A., et al. (Antisense Res. and Develop., 1994, 4, 185-199) describe the use of Dowex-50 in lieu of the G-25 column.
In another method, sodium acetate is used as the salt during HPLC, thereby forgoing the need for recovery of the sodium salt. In this method, glacial acetic acid is added directly to the pooled fractions. After reacting for 30 minutes, the oligonucleotide is ethanol precipitated, reconstituted in water and further reacted in glacial acetic acid several additional times to obtain complete removal of DMTr. Then, the oligonucleotide is subjected to a final ethanol precipitation to obtain the purified product.
During oligonucleotide detritylation, a fine balance between detritylation and depurination exists. Incubating the oligonucleotide in acid for too short of a time will results in incomplete detritylation, while too long of a time will result in increased depurination, thereby reducing yields and purity. In small-scale syntheses, the yield is not an important consideration, due to the small amounts required for typical uses. For the large-scale synthesis of oligonucleotides, each step needs to be optimized to achieve maximum yields.
In the art, it is recognized that optimizing deprotection during oligonucleotide synthesis is a significant problem. WO 96/03417 describes improved methods of detritylation during oligonucleotide synthesis. Paul, C. H., and Royappa, A. T. (Nucleic Acids Res., 1996, 24,3048-3052) and Septak, M. (Nucleic Acids Res., 1996,24,3053-3058) also describes ways of optimizing detritylation during oligonucleotide synthesis.
Yet, optimization of deprotection post-oligonucleotide synthesis is often overlooked. If an attempt is made to achieve maximum post-synthesis deprotection, the incubation time for each oligonucleotide is determined empirically as deprotection rates are specific to an oligonucleotide sequence and particular reaction conditions.
Thus there is the need for improved methods to optimize deprotection of a acid-labile protecting group-containing oligonucleotide following synthesis.